Adoptive cell transfer therapy (ACT) is a promising cancer treatment option. In this regimen, tumor-reactive T cells are cultured ex vivo and infused into a lymphodepleted patient. Priming CD8+ tumor-reactive T cells in the presence of IL-12 polarizes cells to a Tc1 phenotype. These IL-12-primed CD8+ T cells exhibit dramatically enhanced anti-tumor activity when administered in combination with lymphodepletion, though the mechanism behind this synergy is unknown. Established benefits of lymphodepletion include elimination of regulatory T cells (Tregs) and increased availability of IL-2, IL-7 and IL-1, which are important for T cell proliferation and function. Our lab has found that Tc1 cells have enhanced responsiveness to these cytokines in vitro, and they demonstrate dependence on these cytokines in vivo. Also, we have determined that Tc1 cells transferred into an irradiated host dampen the recovery of Tregs. These findings led us to hypothesize that Tc1 cells synergize with the post-lymphodepletion cytokine milieu to create an environment more conducive to the generation of effective anti-tumor immunity. To test this hypothesis 3 aims will be assessed. First, the requirement of homeostatic cytokines for the persistence and anti-tumor efficacy of these IL-12 primed (Tc1) cells will be evaluated using a combination of knockout mice, neutralizing antibodies and cytokine supplementation. Next, we will determine the mechanism of the observed Tc1-mediated reduction in host Tregs post-lymphodepletion and its physiological relevance to Tc1's potent anti-tumor immunity. Finally, we will validate our results using human CD8+ T cells. The first 2 aims will test this hypothesis in the pmel T cell receptor (TCR) transgenic mouse model, and the third will use ACT of human TCR transduced T cells into NSG immunocompromised mice. This transduction protocol is currently part of a Phase I clinical trial being conducted by our collaborators. Successful completion of our aims will provide a directly translatable result that will enable our translational research group to initiate a 2nd rial incorporating IL-12 priming of T cells.